We have previously described pluripotent, parity-induced mammary epithelial cells (PI-MEC) marked by Rosa26-lacZ expression that survives involution in mammary glands of parous females. The PI-MEC appear to represent lobule-limited epithelial stem/progenitor cells. To determine whether pregnancy and secretory differentiation was necessary to generate PI-MEC, we incubated mammary explant cultures from virgin mice in vitro with insulin alone (I), hydrocortisone alone (H), prolactin alone(Prl) or a combination of these lactogenic hormones (IHPrl). Insulin by itself was able to activate the WAP-Cre gene. Hydrocortisone and prolactin alone did not. Any combination of hormones that included insulin was effective. Only the three hormones together (IHPrl) were able to induce secretory differentiation and milk protein synthesis. In addition EGF, IGF-1 or IGF-2 also activated PI-MEC in explant cultures. Neither estrogen nor progesterone brought about WAP-Cre expression. None of these positive initiators of WAP-Cre expression in PI-MEC were effective in mammospheres or two-dimensional cultures of mammary epithelium. This indicates the indispensability of epithelial-stromal interaction in PI-MEC activation. Like PI-MEC, lacZ-positive cells in the treated explants were capable of proliferation and self-renewal both in vitro in mammosphere cultures and in vivo after transplantation into epithelium-divested mammary fat pads. In outgrowths developed in pregnant hosts, lacZ-positive cells were present in mammary alveolar structures as well as ducts and contributed both myoepithelial and luminal progeny. These data demonstrate the presence of a mammary epithelial sub-population in nulliparous female mice before parity that is capable of proliferation, self-renewal and the capacity to produce progeny of diverse cell fates. Previous studies have indicated that pluripotent, parity-identified mammary epithelial stem/progenitor cells (PI-MEC) may be the targets for neoplastic transformation by MMTV-LTR driven neu expression. If this conclusion is correct then MMTV-neu-transformed PI-MEC may represent tumorigenic stem/progenitor cells responsible for the initiation and maintenance of MMTV-neu-induced mammary tumors. To test this hypothesis, MMTV-neu-induced mammary tumor cells were mixed with normal mammary epithelial cells in a dilution series and inoculated into gland-free mammary fat pads. At higher dilutions, MMTV-neu-induced tumor cells interacted with the normal cells to form normal chimeric outgrowths. The tumor cells, lineally marked by lacZ expression, contributed both luminal and myoepithelial progeny to the chimeric gland. LacZ-positive progeny within the chimeric glands continued to express neu and remained negative for estrogen and progesterone receptors. Nevertheless, they contributed to second transplant outgrowths and produced progeny that expressed milk proteins in pregnant hosts. Cultured cells from the secondary outgrowths were positive for MMTV-neu and beta-galactosidase expression but even 5 X 105 cells from these cultures were unable to form tumors (or normal outgrowths) when inoculated into cleared mammary fat pads. Evaluation of both the original tumor cells and those recovered from secondary outgrowths showed high levels for the expression of markers currently associated with mammary stem/progenitor cells (CD49f, CD29 and CD24). These data suggest that the tumorigenic phenotype of MMTV-neu transformed cells can be suppressed by interaction with the normal mammary microenvironment in an expanding epithelial population and lend support the conclusion that MMTV-neu-induced mammary tumors may be derived from MMTV-neu-tranformed PI-MEC. In the last report, we characterized a parity-induced mammary epithelial cell population, which possessed the properties of pluripotency and self-renewal upon transplantation. These cells were lineally marked by the expression of beta-galactosidase (LacZ) as a result of mammary-specific activation of a reporter gene through cre-lox recombination during pregnancy. We used this experimental model to determine if testicular cells would alter their cell fate upon interaction with the mammary gland microenvironment during pregnancy, lactation and involution. Adult testicular cells, isolated from seminiferous tubules, were mixed with limiting dilutions of dispersed mammary epithelial cells and injected into epithelium-divested mammary fat pads. The host mice were bred 6-8 weeks later and examined 20-30 days post involution. This approach allowed for the growth of mammary tissue from the injected cells, transient activation of the WAP (whey acidic protein)-Cre gene during pregnancy and lactation leading to Cre-lox recombination and constitutive expression of LacZ from its promoter. Here we show that cells from adult seminiferous tubules interact with mammary epithelial cells during regeneration of the gland. They adopt mammary epithelial progenitor cell properties including self-renewal and the production of cell progeny, which differentiate into functional mammary epithelial cells. Our results provide evidence for the ascendancy of the tissue microenvironment over the intrinsic nature of cells from an alternative adult tissue.